CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines

Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP-2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern.     

Isolation of the CXC chemokines ENA-78, GRO alpha and GRO gamma from tumor cells and leukocytes reveals NH2-terminal heterogeneity. Functional comparison of different natural isoforms.

Chemokines are a family of chemotactic peptides affecting leukocyte migration during the inflammatory response. Post-translational modification of chemokines has been shown to affect their biological potency. Here, the isolation and identification of natural isoforms of the neutrophil chemoattractants GRO alpha and GRO gamma and the epithelial-cell-derived neutrophil attractant-78 (ENA-78), is reported. Cultured tumor cells produced predominantly intact chemokine forms, whereas peripheral blood monocytes secreted mainly NH2-terminally truncated forms. The order of neutrophil chemotactic potency of these CXC chemokines was GRO alpha > GRO gamma > ENA-78 both for intact and truncated forms. However, truncated GRO alpha (4,5,6-73), GRO gamma (5-73) and ENA-78(8,9-78) were 30-fold, fivefold and threefold more active than the corresponding intact chemokine. As a consequence, truncated GRO alpha (4,5,6-73) was 300-fold more potent than intact ENA-78 indicating that both the type of chemokine and its mode of processing determine the chemotactic potency. Similar observations were made when intact and truncated GRO alpha, GRO gamma and ENA-78 were compared for their capacity to induce an increase in the intracellular calcium concentration in neutrophilic granulocytes, and to desensitize the calcium response towards the CXC chemokine granulocyte chemotactic protein-2 (GCP-2). It must be concluded that physiological proteolytic cleavage of CXC chemokines in general enhances the inflammatory response, whereas for CC chemokines NH2-terminal processing mostly results in reduced chemotactic potency.     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

Hair bundles are specialized for ATP delivery via creatine kinase

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.     

Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family.

The MAGUKs (membrane-associated guanylate kinase homologs) are a family of proteins that act as molecular scaffolds for signaling pathway components at the plasma membrane of animal cells. They are localized in and required for the formation of several types of cell junctions, including epithelial tight and septate junctions as well as synaptic and neuromuscular junctions. They are also localized at the plasma membrane of other cell types, including erythrocytes, where they contribute to cell shape maintenance. MAGUKs function mainly by binding directly to the cytoplasmic termini of transmembrane proteins as well as to other signal transduction proteins. They appear to hold together elements of individual signaling pathways, thereby contributing to the efficiency and specificity of signaling interactions while simultaneously maintaining the structural specializations of the plasma membrane. BioEssays 1999;21:912-921.     

When specialized sites are important for synapsis and the distribution of crossovers

In C. elegans and D. melanogaster, specialized sites have an important role in meiotic recombination. Recent evidence has shown that these sites in C. elegans have a role in synapsis. Here we compare the initiation of synapsis in organisms with specialized sites and those without. We propose that, early in prophase, synapsis requires an initiator to overcome inhibitory factors that function to prevent synaptonemal complex (SC) formation between nonhomologous sequences. These initiators of SC formation can be stimulated by crossover sites, possibly other types of recombination sites and also specialized sites where recombination does not occur.     

Human monocyte or recombinant interleukin 1's are specific for the secretion of a metalloproteinase from chondrocytes

Macrophage products induce production of proteases that contribute to cartilage degradation in various joint diseases. In these studies we stimulated rabbit chondrocytes with various cytokines in vitro in order to determine which were responsible for changes in the release of prostaglandin, plasminogen activator, and a metalloproteinase. The metalloproteinase assayed in these studies is a latent enzyme whose activity can rapidly be measured with fluorogenic casein. Conditioned media from stimulated human peripheral blood mononuclear cells; purified human monocyte IL 1, pI 7,6, and 5; and recombinant human IL 1, beta or alpha forms, all changed the secretory pattern of rabbit articular chondrocytes in a similar manner: production and secretion of a latent metalloproteinase(s) and prostaglandin E were stimulated in a concentration-dependent fashion, whereas the activity of plasminogen activator was strongly reduced. Antibodies against human monocyte IL 1 blocked the active principle in various mononuclear cell-conditioned media, suggesting that uncharacterized factors present in these supernatants do not affect the metalloproteinase response. When added to confluent chondrocytes, phorbol myristate acetate, concanavalin A, IL 2, lipopolysaccharide, indomethacin, and prostaglandin E2, which interfere with lymphocyte proliferation assays for IL 1, failed to influence chondrocyte metalloproteinase secretion. Recombinant human IFN-alpha or IFN-gamma in the presence or absence of IL 1 had no effect on rabbit chondrocytes, whereas recombinant human tumor necrosis factor decreased plasminogen activator but had no effect on prostaglandin or metalloproteinase production. These results support the concept that IL 1 specifically induces chondrocytes to produce metalloproteinases, and hence may play an important role in destructive joint diseases.     

S-phase cyclins are required for a stable arrest at metaphase

A critical DNA damage checkpoint in Saccharomyces cerevisiae is an arrest at the metaphase stage of mitosis. Here we show that the S-phase cyclins Clb5 and Clb6 are required for this arrest. Strains lacking Clb5 and Clb6 are hypersensitive to DNA damage. Furthermore, in the presence of the DNA alkylating agent methyl methanesulfonate (MMS) over 50% of clb5 clb6 mutants by-passed the metaphase checkpoint and arrested instead with separated sister chromatids. Levels of Pds1, an inhibitor of anaphase that accumulates following DNA damage, were similar in the wild-type and mutant strains following MMS treatment. Furthermore, unlike wild-type cells, clb5 clb6 mutants undergo nuclear division despite the presence of nuclear non-degradable Pds1. Our results suggest a novel role for the S-phase cyclins Clb5 and Clb6 in maintaining sister chromatid cohesion during a metaphase arrest, perhaps by regulating Pds1 activity.     

Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.     

Why children from the same family are so different from one another

The well-established finding that siblings growing up in the same family turn out to be very different from one another has puzzled psychologists and behavior geneticists alike. In this theoretical note we describe the possible ontogeny and phylogeny of a sibling differentiation mechanism. We suggest that sibling competition for parental investment results in sibling differentiation on a number of characteristics, producing different developmental trajectories within families. Variations in developmental trajectories within families may have had fitness advantages in ancestral environments because(a) sibling competition for extrafamilial resources would be reduced and(b) these variations would be suited to environments containing a variety of niches or to changing environments. Predictions derived from this model and an example of an application to attachment theory are presented.     

Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi

In Saccharomyces cerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the trans-Golgi network (TGN)/early endosome to the plasma membrane requires the function of Chs5p and Chs6p. Chs6p belongs to a family of four proteins that we have named ChAPs for Chs5p-Arf1p-binding Proteins. Here, we show that all ChAPs physically interact not only with Chs5p but also with the small GTPase Arf1p. A short sequence at the C-terminus of the ChAPs is required for protein function and the ability to bind to Chs5p. Simultaneous disruption of two members, Deltabud7 and Deltabch1, phenocopies a Deltachs6 or Deltachs5 deletion with respect to Chs3p transport. Moreover, the ChAPs interact with each other and can form complexes. In addition, they are all at least partially localized to the TGN in a Chs5p-dependent manner. Most importantly, several ChAPs can interact physically with Chs3p. We propose that the ChAPs facilitate export of cargo out of the Golgi.     

Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest

Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwarf-derived fibroblasts are slow to enter the stage of growth arrest induced by culturing normal cells under standard culture conditions at 20% O(2). Dwarf cells cultured at 20% O(2) resemble control cells cultured at 3% O(2) not only in their delayed growth arrest, but also in their rapid growth rates and resistance to both oxidative and nonoxidative forms of cytotoxic stress. Levels of the heat-shock protein HSP-70 respond to serum withdrawal and stress only in control cells, showing that intracellular signals are blunted in dwarf-derived cells. These data suggest a model in which stable epigenetic changes induced in skin fibroblasts by the hormonal milieu of the Snell dwarf lead to resistance to multiple forms of injury, including the oxidative damage that contributes to growth arrest in vitro and neoplasia in intact mice.     

Bcl-2 family proteins are essential for platelet survival

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.     

How reproducible are flow cytometry data from paraffin-embedded blocks?

The purpose of this technical report is to determine the reproducibility of flow cytometry data for ploidy and cell cycle kinetics using paraffin-embedded blocks of breast cancer tissue. One block from each of 39 tumors was studied in this report with each block having multiple sections analyzed independently. All of these sections gave ploidy analyses, while only 34 gave cell kinetic values. The standard deviation for the DNA index value in the multiple analysis study was less than 0.1 in all but three cases. The cell kinetic values gave larger variability, and the actual values were dependent on the method of analysis. Comparison of the variability for each method of analysis could not predict which procedure was superior. These results would indicate that ploidy is a reproducible value, while cell kinetic parameters should only be used as an indicator of proliferative activity that has been normalized to the mean or median of a large set of observations processed and analyzed by the same procedure.     

Villous B cells of the small intestine are specialized for invariant NK T cell dependence

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM(+) IgD(+) cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinase(xid), Galphai2(-/-)), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jalpha18(-/-) or CD1d(-/-) mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.     

Equilibrative nucleoside transporter family members from Leishmania donovani are electrogenic proton symporters

Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters.     

Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila

Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. We find that autophagy is induced following growth arrest and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. Our results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death and that multiple degradation pathways cooperate in the efficient clearance of cells during development.